Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on PGP 2000 column (0.10 M sodium phosphate buffer, 0.30 M NaCl, pH 6.86) showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the second one to human nonmercaptalbumin (HNA). Mechanism for the separation of HMA and HNA might be due to weak resin-HSA interaction. HPLC analysis of bovine plasma albumin (BPA) showed a single peak on PGP 2000 column. The elution volume of HSA was larger than that of BPA, resulting in a clear resolution of HSA and BPA.     

HPLC-studies on nonmercapt-mercapt conversion of human serum albumin

Human mercaptalbumin (HMA) and nonmercaptalbumin (HNA) could be separated by high-performance liquid chromatography (HPLC) at neutral pH. Using HPLC, the present authors found the nonmercapt-mercapt conversion (HNA ← HMA) during hemodialysis and the mercapt-nonmercapt conversion (HMA ← HNA) after hemodialysis in chronic renal failure, indicating HMA as the covalent carrier protein for sulfur-containing amino acids.     

反相高效液相色谱法测定人血浆中甲磺丁脲的含量

目的建立反相高效液相色谱法用于测定人血浆中甲磺丁脲的含量。方法采用Eclipse XDB-C_(18)色谱柱,以乙腈-25 mmol·L~(-1)乙酸钠缓冲液(pH=3.3,32:68)为流动相,流速1.0 mL·min~(-1),检测波长为229 nm,柱温35℃。血样经等体积比的0.6 mol·L~(-1)三氯乙酸处理后,离心,取上清液20μL,进样检测。结果甲磺丁脲血浆药物浓度在0.5～100 mg·L~(-1)内,线性关系良好(r=0.999 6);回收率为93.0%～105.0%;日内RSD≤3.80%,日间RSD≤6.31%。结论本方法简单快速、准确灵敏、回收率高、重现性好,适用于甲磺丁脲的血药浓度测定。     

Determination of amoxapine and its metabolites in human serum by high-performance liquid chromatography

The simultaneous quantitative determination of amoxapine, 7-hydroxyamoxapine and 8-hydroxyamoxapine in human serum was established, with good recoveries, using reversed-phase high-performance liquid chromatography (HPLC). Prior to analysis by high-performance liquid chromatography, the enzymic hydrolysis with β-glucuronidase/arylsulphatase of sera from healthy volunteers receiving the drug snowed that each conjugate of two hydroxyamoxapines was 75–90% of the amount determined by the present method. The concentrations of amoxapine and its hydroxylated metabolites were measured against time in sera from the volunteers who were given the antidepressant orally for 2 weeks. The serum levels of 8-OH-amoxapine were markedly higher than the drug itself and the 7-OH-derivative. Whereas the levels of the drug were little increased during the continuous administration, the levels of 8-OH-amoxapine were linearly increased until the fourth day after the administration was started. In addition, the ratio of each hydroxylated metabolite to the drug and the time-course of their serum levels varied interindividually.     

Fatty acid and drug binding to a low-affinity component of human serum albumin,purified by affinity chromatography

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

透析-色谱联用技术研究黄连生物碱与牛血清白蛋白的结合作用

目的:研究黄连中与牛血清白蛋白(BSA)有结合作用的潜在活性成分。方法:采用平衡透析与高效液相色谱联用技术筛选黄连中与BSA有结合作用的成分;考察BSA浓度、缓冲液pH值对黄连生物碱与BSA结合作用的影响;比较黄连中各生物碱单体与BSA单独作用和黄连总生物碱与BSA作用的区别。结果:黄连中共有7个化学成分与BSA结合作用明显,通过对照品对照,其中6个成分被分别鉴定为非洲防己碱、药根碱、表小檗碱、黄连碱、巴马亭及盐酸小檗碱;各生物碱单独结合作用强于它们在黄连中混合存在的结合作用,各生物碱在与BSA结合过程中存在竞争作用。结论:蛋白结合-平衡透析-高效液相色谱法联用可有效、快速地预测黄连中多种成分在体内的吸收情况,筛选其潜在活性成分,且能体现中药整体作用的特点,为其药效物质基础的进一步研究提供依据。     

Simultaneous determination of codeine and ibuprofen in plasma by high-performance liquid chromatography

A procedure is described for the simultaneous determination of codeine and ibuprofen in human plasma following the administration of the two substances in a proposed combination dosage form. The two substances were extracted separately from plasma and then determined together by high-performance liquid chromatography (HPLC) using a fluorescence detector. The codeine was first extracted from alkalinized plasma with hexane-dichloromethane (2:1, v/v) and then washed with sodium hydroxide solution. The ibuprofen was then extracted with hexane from the plasma acidified with sulphuric acid. The organic layers were collected, evaporated to dryness and the reconstituted residue was subjected to HPLC. The detection limit for codeine was 8 microg 1(-1) and for ibuprofen 1 mg 1(-1).     

Quantitative analysis of demeclocycline by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method suitable for the quality control of demeclocycline is described. The stationary phase is a poly(styrene-divinylbenzene) copolymer, kept at 60°C. The mobile phase comprises 2-methyl-2-propanol−0.2 M potassium phosphate buffer (pH 9.0)−0.02 M tetrabutylammonium hydrogen sulphate (pH 9.0)−0.01 M sodium edetate (pH 9.0)—water (8:10:15:10:57, m/v/v/v/v). The flow rate is 1 ml min⁻¹ and detection is performed at 254 nm. Official standards are compared and results for the analysis of a number of commercial bulk samples and preparations are presented. 4-Epidemeclocycline and demethyltetracycline are the main impurities. 4-Epidemethyltetracycline and 2-acetyl-2-decarboxamido-demeclocycline can also be present.     

The determination of D-penicillamine and its disulphide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Reversed-phase ion-pair conditions are used for the determination of D-penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.     

反相高效液相色谱法测定大鼠脏器组织中吉西他滨的含量

目的建立反相高效液相色谱法用于测定大鼠胰腺、肝脏、脾脏、肾脏、肺脏、心脏、腹直肌组织中吉西他滨的含量。方法采用phenomenex色谱柱，5-溴尿嘧啶为内标，流动相为乙腈-0．1％三氟乙酸溶液（3：97，V／V），流速1．0mL·min^-1，检测波长268am，柱温45℃。组织样品经甲醇-乙腈（1：9，V／V）蛋白沉淀处理后，55℃恒温水浴中氮气吹干，经流动相溶解后进样，进样量50μL。结果大鼠各脏器组织中吉西他滨在浓度1～100mg·L^-1内线性关系良好，方法回收率为93％～104％，提取回收率〉70％，日内、日间RSD均〈6％。结论本方法操作简单，重现性好，可用于大鼠多种组织中吉西他滨的含量测定。     

高效液相色谱法测定大鼠血浆中的二氯二茂钛

目的 建立反相高效液相色谱法测定大鼠血浆中有机金属抗癌原料药二氯二茂钛的含量.方法 采用Shimadzu VP-ODS色谱柱(250×4.6 mm,5 m),V(甲醇):V(醋酸铵)=55:45,pH 2.5为流动相,流速为1.0 ml/min,紫外检测波长254 nm,柱温室温,氨基比林为内标进行测定.血浆中生物样品采用液-液萃取法进行提取.结果 二氯二茂钛在大鼠血浆中的线性范围为10～120 μg/ml,回归方程为Y=57.83X-0.6042(r=0.9991,n=7),方法精密度RSD为1.8 %(n=6),平均回收率为RSD=1.63%(n=9).结论 方法简单快速,专属性强,可用于测定大鼠血浆中二氯二茂钛的含量,为该类药物的体内血药浓度检测和动力学研究提供了实验依据.     

高效液相色谱法测定血浆中氟哌啶醇浓度

目的：建立高效液相色谱法测定血浆中氟哌啶醇浓度的方法。方法：以乙腈甲醇０ ．０２５ｍｏｌ／Ｌ 磷酸二氢钾（４５∶５∶５０ ,ｖ／ｖ） 为流动相,氯氟哌啶醇作内标,紫外波长２４８ｎｍ 处检测。结果：血浆中氟哌啶醇的最低检测浓度为０ ．５ｎｇ／ｍｌ,平均提取回收率为８６ ．６ ％ ±４ ．９ ％ ,线性范围１ ～５０ｎｇ／ｍｌ（ｒ ＝ ０ ．９９９８） ,日内和日间ＲＳＤ 分别小于８ ％ 和９ ％ 。结论：方法灵敏、快速、准确,可满足临床治疗药物的监测工作     

The simultaneous determination of serum cortisol and cortisone by high-performance liquid chromatography with fluorimetric detection

A new method for the simultaneous determination of cortisol and cortisone in serum by high-performance liquid chromatography with fluorimetric detection has been developed. The two steroids were derivatized by treatment with 9-anthroyl nitrile in triethylamine-acetonitrile to give the fluorescent esters through the 21-hydroxyl group. These derivatives were efficiently separated on a straight-phase Cosmosil 5SL column with hexane-ethyl acetate (6:5) as eluent. The detection limit was 20 fmol, using the 9-anthroyl derivative of prednisolone as an internal standard. The use of Sep-Pak C18 and Clin-Elut cartridges proved to be efficient for the clean-up of cortisol and cortisone in serum samples.     

超高效液相色谱法测定人血清中多索茶碱浓度

目的建立超高效液相色谱法测定人血清中多索茶碱浓度。方法建立色谱条件,色谱柱为Waters Acquity C₁₈柱（50mm×2.1mm,1.7μm）,流动相为乙腈-水（梯度洗脱）,流速为O.2mL·mill^-1,内标为咖啡因,柱温为室温,检测波长为273nm。结果多索茶碱血药浓度在0.18～23.20 mg·L^-1内,与A多索茶碱/A咖啡因之间的线性关系良好,回归方程为Υ=O.181 8χ+0.005 1（r=O.999 9,n=10）;日内、日问精密度好（RSD均≤15%）,方法回收率为94.70%～106.16%。结论该方法快速、灵敏、准确,可用于多索茶碱血药浓度的测定和药动学研究。     

A reversed‐phase high‐performance liquid chromatography method for bovine serum albumin assay in pharmaceutical dosage forms and protein/antigen delivery systems

Bovine serum albumin (BSA) is among the most widely used proteins in protein formulations as well as in the development of novel delivery systems as a typical model for therapeutic/diagnostic proteins and the new versions of vaccines. The development of reliable and easily available assay methods for quantitation of this protein would therefore play a crucial role in these types of studies. A simple gradient reversed‐phase high‐performance liquid chromatography with ultra‐violet detection (HPLC‐UV) method has been developed for quantitation of BSA in dosage forms and protein delivery systems. The method produced linear responses throughout the wide BSA concentration range of 1 to 100 µ g/mL. The average within‐run and between‐run variations of the method within the linear concentration range of BSA were 2.46% and 2.20%, respectively, with accuracies of 104.49% and 104.58% for within‐run and between‐run samples, respectively. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.5 and 1 µg/mL, respectively. The method showed acceptable system suitability indices, which enabled us to use it successfully during our particulate vaccine delivery research project. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of oligosaccharides and monosaccharides in Hakka rice wine by precolumn derivation high-performance liquid chromatography

This article presents a precolumn derivatization procedure with 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent to detect oligosaccharides and monosaccharides in Hakka rice wine. The subsequent separation of the derivatized glucose–PMP also was performed using a mobile phase consisting of the molar ratio of acetonitrile to ammonium acetate buffer (0.1M) of 22:78 at a flow rate of 1.0 mL/min with the column temperature of 35°C, and the pH of ammonium acetate buffer at 5.5. The optimum derivation conditions were as follows: reaction temperature, 70°C; reaction time, 30 minutes; molar ratio of PMP to glucose, 10:1 (v/v); molar ratio of sodium hydroxide to glucose, 3:1 (v/v). The recovery rates were between 93.13% and 102.08% with relative standard deviation of 0.96–2.48%. The established method provides sufficient sensitivity with values of limit of detection of 0.09–0.26 mg/L and limit of quantification of 0.27–0.87 mg/L for determination of oligosaccharides and monosaccharides.     

The determination of nadolol in biological samples using high-performance liquid chromatography

A method has been developed for the determination of nadolol in biological samples by reversed-phase high-performance liquid chromatography with fluorimetric detection. The method has been applied to plasma, serum and urine samples, which are prepared by extraction with diethyl ether-dichloromethane (5:2,v/v), evaporation of the organic solvent, and dissolution of the resultant residue in the chromatographic eluent. The sample is then subjected to chromatography on a C(18)-silica column, with an eluent of water-acetonitrile-triethylamine (800:200:1,v/v) adjusted to pH 3.0 with orthophosphoric acid. A single point external standard is used for quantitation. The working ranges were 1-400 ng/ml for plasma/serum, and 0.1-40 mug/ml for urine, although a detection limit of 0.1 ng/ml appears to be readily attainable. The sample size was 0.5 ml, and for both types of sample the method showed good correlation with a previously published fluorimetric method (for plasma, r = 0.9544, n = 70; for urine, r = 0.9919, n = 35).     

用高效液相色谱法测定兔血清中硫酸庆大霉素的含量

本文以高效液相色谱法测定血清中硫酸庆大霉素的含量。加内标物茴香胺。乙腈沉淀蛋白质等干扰物，室温下进行邻苯二醛衍生化，醋酸乙酯提取后进样，用紫外３３０ｎｍ检测。     

高效液相色谱法测定人血浆中左布比卡因的浓度

目的建立高效液相色谱法测定人血浆中左布比卡因的浓度。方法以罗哌卡因为内标,色谱柱为Hypersil C_(18)柱(200 mm×4.6 mm,5μm),流动相采用0.01 mol·L~(-1)磷酸二氢钾-乙腈(87:13,V:V,pH=3.4),流速2.0 mL·min~(-1),检测波长为210 nm,柱温40℃。结果标准曲线方程为Y=0.310 1 X+ 0.008 9,r=0.999 7,左布比卡因的质量浓度在0.012 5～2 mg·L~(-1)范围内呈良好的线性关系,最低检测限为0.01 mg·L~(-1);方法回收率在96%～105%之间;日间、日内精密度RSD均<5%。结论本方法可用于临床上左布比卡因血药浓度的监测和药动学研究。